Human serum albumin (HSA or HA), a protein of 585 ammo acids (SEQ ID NO: 26), is responsible for a significant proportion of the osmotic pressure of serum and also functions as a carrier of endogenous and exogenous ligands. At present, HSA for clinical use is produced by extraction from human blood. The production of recombinant HA (rHA) in microorganisms has been disclosed in EP 330 451 and EP 361 991.
The role of albumin as a carrier molecule and its inert nature are desirable properties for use as a stabiliser and transporter of polypeptides. The use of albumin as a component of a fusion protein for stabilising other proteins has been disclosed in WO 93/15199, WO 93/15200, and EP 413 622. The use of N-terminal fragments of HSA for fusions to polypeptides has also been disclosed (EP 399 666). Fusion to the said polypeptide is achieved by genetic manipulation, such that the DNA coding for HSA, or a fragment thereof, is joined to the DNA coding for the said polypeptide. A suitable host is then transformed or transfected with the fused nucleotide sequences, so arranged on a suitable plasmid as to express a fusion polypeptide. Nomura et al (1995) attempted to express human apolipoprotein E in S. cerevisiae as a fusion protein with HSA or fragments of HSA, using the HSA pre-sequence to direct secretion. Whilst fusion to full length HSA resulted in the secretion of low levels of the protein into the medium (maximum yield of 6.3 mg per litre), fusion to HSA (1-198) or HSA (1-390) did not result in secretion into the medium.
Human growth hormone (reviewed by Strobl and Thomas, 1994) consists of a single polypeptide of 191 amino acids, internally cross-linked by two disulphide bonds. Two molecules of hGH receptor bind each molecule of hGH to facilitate signal transduction (Cunningham et al, 1991; de Vos et al, 1992). The C-terminus of the hGH molecule is involved in binding to the first receptor molecule, but the extent to which the N-terminus is involved in receptor binding is not known. The hormone is secreted from the anterior pituitary gland under hypothalamic control, and is responsible for a wide range of growth-promoting effects in the body. Clinically, hGH is used in the treatment of hypopituitary dwarfism, chronic renal insufficiency in childhood, bone fractures and burns. Current methods of production of hGH for therapeutic use are by extraction from human pituitary gland, recombinant expression in Escherichia coli as disclosed in EP 127 305 (Genentech) or recombinant expression in mammalian cell culture (Zeisel et al, 1992).
In addition, hGH has been expressed intracellularly in yeast (Tokunaga et al, 1985) and this organism may provide an alternative means of production as disclosed in EP 60 057 (Genentech). Tsiomenko et al (1994) reported the role of the yeast MFα-1 prepro leader sequence in the secretion of hGH from yeast. Attachment of the pre-portion of the leader sequence to the hGH gene resulted in hGH accumulation in the periplasm and vacuoles, whilst attachment of the pro-portion to hGH resulted in expression of a non-glycosylated precursor localised inside the cell. Only when both portions of the leader sequence were attached to the hGH gene was hGH secreted into the culture medium. Other secretion signals (pre-sequences) were also ineffective unless a yeast-derived pro sequence was used, suggesting that such a pro sequence was used is critical to the efficient secretion of hGH in yeast.
In humans, hGH is secreted into the blood in pulses, and in the circulation has a half-life of less than 20 minutes (Haffner et al, 1994). Elimination of the hormone is primarily via metabolism in the liver and kidneys and is more rapid in adults than in children (Kearns et al, 1991). Treatment for hGH deficiency generally lasts for 6 to 24 months, during which hGH is administered either three times a week intramuscularly or on a daily basis subcutaneously. Such a regimen of frequent administration is necessary because of the short half-life of the molecule.
Poznansky et al (1988) increased the half-life of porcine growth hormone by conjugation with either porcine or human serum albumin (HSA) to form relatively large conjugates of about 180 kD. Chemical reaction using the cross-linking reagent glutaraldehyde resulted in, on average, two molecules of albumin complexed with six molecules of growth hormone. The resulting 180 kD conjugate was found to have an extended half-life in the circulation of rats of 2 to 3 hours, compared to 5 minutes for unconjugated growth hormone. Activity assays showed that the conjugate retained full, and possibly increased activity in vitro, but was inactive in vivo.